AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke

We're exploring AAV-mediated ectopic expression of neurogenic transcription factors as a potential treatment for ischemic stroke. Our focus is to see if Neurod1 expression in GFAP-expressing cells during the subacute and chronic phases after a stroke increases transduced neurons and correlates with motor function improvements. Recent findings show that gene expression in the cortex varies by region with the same AAV Our research found that the GFAP promoter is more active in medial prefrontal cortex than in the motor cortex, indicating that transduced cells respond differently to specific promoters.

Thus, therapies involving neurogenic transcription factors should be tailored to the target brain region. Current challenges include variability in stroke lesion size due to variables such as the injection rates of ET-1 and transduction efficiencies between brain regions. AAV titers and tropism across brain regions and mouse strains can affect transduction efficiencies, impacting the cellular assessment of ectopic Neurod1 expression post stroke.

The main challenge in converting astrocyte to neurons is distinguishing reprogrammed neurons from preexisting ones. Our lab aims to confirm that this reprogramming occurs in vivo by developing novel tools. Ultimately, we seek to understand how ectopic expression of neuronal transcription factor enhances recovery in brain-injured animals.

To begin, remove the anesthetized animal from the chamber and place it onto a clean surface. After cleaning the surgical area of the mouse, place its nose in the stereotaxic nose cone and stabilize the skull using ear bars. Using a number 10 scalpel blade, make a vertical incision parallel to the sagittal plane from behind the midpoint of the eyes to the parietal bone.

Then, gently retract the skin to expose the skull. Next, use a Q-tip to disrupt the fascia on the skull and allow the skull to dry to fully expose the bregma. Using a high-speed stereotaxic drill, drill three burr holes at the coordinates in the right sensory motor cortex.

Then, replace the drill with a 26 gauge syringe equipped with a needle measuring 0.375 inches in length. Load 1.5 microliters of 400 micromolar endothelin-1 solution dissolved in sterile PBS into the syringe. Release a small volume of endothelin-1 to observe a drop at the tip of the needle, ensuring good flow.

Lower the tip of the needle past the skull into the middle burr hole without puncturing the dura mater. Once aligned, lower the needle one millimeter into the brain. Using the Hamilton syringe, dispense 0.1 microliter at a time, and wait for one minute before injecting the next 0.1 microliter.

To prevent the needle from becoming blocked between and after injections, release a small volume of endothelin-1 until a drop appears at the needle tip. Using a sterile Q-tip, wipe off the excess endothelin-1 before injection. Finally, suture the overlying skin on the skull using a 4-0 sterile suture to close the wound.

After anesthetizing the endothelin-1-treated mouse, transfer the mouse to the stereotaxic frame. Using surgical scissors, cut the sutures on the wound. With forceps, remove the scab and retract the skin to expose the skull.

Then, use a sterile Q-tip to dry the skull surface and locate the burr holes. Set up the manipulator arms of the stereotaxic apparatus. Place a needle holder carrying a 26 gauge syringe with a needle measuring 0.375 inches in length.

Now, load 3.4 microliters of AAV solution into the syringe. Lower the tip of the needle past the skull into the first, most anterior burr hole. Ensure the needle does not puncture the dura mater, then lower at one millimeter into the brain.

Using the Hamilton syringe, inject one microliter of AAV solution by dispensing 0.1 microliter at a time as demonstrated earlier. Then suture the overlying skin on the skull to close the wound using a 4-0 Polysorb polyester suture. Remove the mouse from the stereotaxic frame and transfer it to a clean, preheated animal housing cage in the recovery area.